Obesity and insulin resistance afflict millions of Americans. Data from the National Health and Nutrition Survey, cycle III 1988-94, (NHANES III) suggests that there is in excess of 23% of Americans who are obese (Kuczmarski et al., Obes. Res. 5:542-548, 1997). At least a similar number of individuals are known to be overweight. This large epidemic results in increased mortality and morbidity from cardiovascular disease and Type 2 diabetes (Grundy et al., Circulation 110:227-239, 2004). Among emerging co-morbidities of obesity and insulin resistance is a condition called nonalcoholic fatty liver disease (NAFLD) (Liangpunsakul et al., Curr. Treat. Options Gastroenterol. 6:455-463, 2003). Nonalcoholic fatty liver disease is characterized by macrovesicular steatosis of the liver occurring in individuals who consume little or no alcohol. The histological spectrum of NAFLD includes the presence of steatosis alone, fatty liver and inflammation and a more aggressive form called nonalcoholic steatohepatitis (NASH), characterized by the presence of steatosis, inflammation and varying degrees of fibrosis (Brunt et al., Am. J. Gastroenterol. 94:2467-2474, 1999; Brunt, Semin. Liver Dis. 21:3-16, 2001). NASH can progress into cirrhosis and permanent liver failure (Brunt et al., supra, 1999; Brunt, supra, 2001; Ludwig et al., J. Gastroenterol. Hepatol. 12:398-403, 1997).
The lack of symptoms in the early stages and the absence of uniform diagnostic criteria make it difficult to determine the true prevalence of NAFLD and its various forms. Data from an autopsy study in Canada found that roughly 20% of obese individuals have hepatic steatosis (Scheen et al., Best Pract. Res. Clin. Endocrinol. Metab. 16:703-716, 2002). More recently, a landmark study of NMR spectroscopy carried out on the population of the Dallas Heart study, indicated that more than a third of the study population, a sedentary urban United States population comprising 2,349 patients with an excellent representation of different sexes, races and ethnic groups, had hepatic steatosis (Browning et al., Hepatology 40:1387-1395, 2004). The rate of observing the aggressive form NASH is not known in this population, but about ⅕ of these patients are predicted to have NASH if they were subjected to a biopsy (McCullough, J. Clin. Gastroenterol. 34:255-262, 2002). Thus, NASH is estimated to be prevalent at a rate of at least 5% of the general population.
Nonalcoholic steatohepatitis (NASH) lies on a spectrum of nonalcoholic fatty liver disease (NAFLD) that ranges from ectopic lipid accumulation (steatosis) to cirrhosis (Matteoni et al., Gastroenterol. 116:1413-1419, 1999). Steatosis is believed to sensitize the liver to metabolic injury leading to inflammation, necrosis, and fibrosis (James et al., Lancet 353:1634-1636, 1999; Ludwig et al., Mayo Clin. Proc. 55:434-438, 1980; Day, Gut 50:585-588, 2002; Browning et al., J. Clin. Invest. 114:147-152, 2004). Thus, steatosis is a constant feature of NASH, but NASH is only distinguishable by liver biopsy. The assessment and severity of NASH is made histologically based on the patterns and degrees of hepatic steatosis, inflammation, and injury and, by definition, occurs only in the absence of significant alcohol consumption (Brunt, supra, 2001). While steatosis is seen in both animal and human models, NASH is only fully appreciated in the human condition (Browning et al., supra, 2004). Thus, understanding the clinical variation observed in NASH is critical for the development of therapeutic strategies for this condition.
NASH is now accepted as a progressive metabolic liver disease. Hepatic fatty infiltration or steatosis is thought to be the precursor for NASH or the “first hit” to the liver in the development of this metabolic liver disease. Steatosis is believed to sensitize the liver to subsequent metabolic injury leading to inflammation, necrosis, and fibrosis (James et al., supra, 1999; Ludwig et al., supra, 1980; Day, supra, 2002; Browning et al., supra, 2004). The liver plays a key role in both glucose and lipid metabolism. Several important studies suggested that insulin resistance was a key feature of hepatic steatosis (de Knegt, Scand. J. Gastroenterol. Suppl.: 88-92, 2001). The engorgement of the liver with lipid causes severe insulin resistance in the liver and abnormal glucose production (Samuel et al., J. Biol. Chem. 279:32345-32353, 2004). Studies from rodents suggest that genes important for adipocyte differentiation (such as peroxisome proliferator-activated receptor gamma 1 (PPARg1), PPARg2, and SREBP-1) are up-regulated in steatotic livers (Brown et al., Nutr. Rev. 56:S1-S3; S54-S75, 1998; Matsusue et al., J. Clin. Invest. 111:737-747, 2003; Yu et al., J. Biol. Chem. 278:498-505, 2003). Furthermore, the presence of peripheral insulin resistance and hyperinsulinemia worsens the problem of increased lipid deposition in the liver, leading to a vicious metabolic cycle (Shimomura et al., Mol. Cell. 6:77-86, 2000). Hence, therapeutic interventions that decrease lipid deposition in the liver are likely to improve both insulin resistance and abnormal hepatic glucose production.
Data has been accumulating to support a “second hit” theory at play on top of lipid deposition in the pathogenesis of NASH (Diehl, Semin. Liver Dis. 19:221-229, 1999). The suspected second hit may be intracellular oxidative stress that can be induced by multiple mechanisms such as excess iron accumulation, endotoxin exposure, pro-inflammatory cytokines or other unknown factors. Accumulation of fatty acids and change of the balance of fatty acids may also be responsible for generation of increased free radicals by being a substrate for lipid peroxidation (de Almeida et al., Clin. Nutr. 21:219-223, 2002).
There is no standard therapy for management of NASH at this point (Agrawal et al., J. Clin. Gastroenterol. 35:253-261, 2002). The first line of medical intervention in obese patients is recommendation of weight loss and exercise (Nehra et al., Dig. Dis. Sci. 46:2347-2352, 2001). Short-term weight loss has been shown to improve insulin resistance, decrease visceral fat deposition and hepatic steatosis; however, its ultimate effect on histological improvement and the natural course of NASH is not known (Petersen et al., Diabetes 54:603-608, 2005). However, long-term adherence and success of this recommendation is questionable. Sibutramine and Orlistat have been reportedly associated with improved sonographic findings and insulin resistance (Sabuncu et al., Rom. J. Gastroenterol. 12:189-192, 2003). The most promising drugs thus far have been insulin sensitizers, like metformin (Lin et al., Nat. Med. 6:998-1003, 2000; Marchesini et al., Lancet 358:893-894, 2001) and thiazolidinediones rosiglitazone (Neuschwander-Tetri et al., Hepatology 38:1008-1017, 2003; Neuschwander-Tetri et al., J. Hepatol. 38:434-440, 2003. and pioglitazone (Shadid et al., Clin. Gastroenterol. Hepatol. 1:384-387, 2003; Promrat et al., Hepatology 39:188-196, 2004). Despite the encouraging results observed with these drugs, the need for additional new therapies is evident.
In rodent models, steatosis occurs with decreased leptin action, whether due to leptin deficiency or resistance (Lee et al., Proc. Natl. Acad. Sci. U.S.A. 99:11848-11853, 2002; Unger, Diabetes 44:863-870, 1995; Unger et al., Proc. Natl. Acad. Sci. U.S.A. 96:2327-2332, 1999; Wang et al., J. Biol. Chem. 274:17541-17544, 1999). Rodent models of leptin deficiency either have mutations in the ob gene encoding leptin (the ob/ob mouse) or they lack fat, the organ manufacturing leptin. Animal models lacking white adipose tissue have been engineered using a couple of different strategies. Regardless of the strategy, the deficiency of white adipose tissue leads to leptin deficiency, insulin resistance, severe hypertriglyceridemia and massive hepatic steatosis (Reitman et al., Trends Endocrinol. Metab. 11:410-416, 2000). Evaluation of insulin sensitivity in one of these models using a hyperinsulinemic euglycemic clamp demonstrated decreased hepatic insulin sensitivity, i.e. dysregulated hapatic glucose production (Kim et al., J. Biol. Chem. 275:8456-8460, 2000). Transplantation of fat from littermates, into this model resulted in amelioration of total body insulin sensitivity, dyslipidemia, hepatic steatosis and also hepatic glucose production in a dose dependent manner (Reitman et al., Trends Endocrinol. Metab. 11:410-416, 2000; Kim et al., J. Biol. Chem. 275:8456-8460, 2000, Gavrilova et al., J. Clin. Invest. 105:271-278, 2000). In contrast, if fat is procured from ob mice, the metabolic amelioration of fat transplantation is not observed (Colombo et al., Diabetes 51:2727-2733, 2002).
Shimomura et al, tested whether replacing leptin made an impact on the insulin sensitivity, dyslipidemia and hepatic steatosis in a model of fat deficiency. A major impact of physiological leptin replacement was evident after 3 weeks of therapy (Shimomura et al., Genes Dev. 12:3182-3194, 1998; Shimomura et al., Nature 401:73-76, 1999). Furthermore, this study showed that lipogenesis was dysregulated in the liver in the absence of leptin which was corrected with leptin replacement. Other reports confirmed the validity of these observations in other models. The observations related to the efficacy of leptin in the fatless mice formed the basis for our testing the efficacy of leptin replacement therapy in a human disease characterized by absence of body fat, namely lipodystrophy (Oral et al., N. Engl. J. Med. 346:570-578, 2002). In addition to displaying severe insulin resistance, dyslipidemia and hepatic steatosis, patients with generalized lipodystrophy also acquire NASH. In fact, cirrhosis and liver failure are important causes of premature death in these patients (Taylor et al., J. Basic Clin. Physiol. Pharmacol. 9:419-439, 1998; Oral, Rev. Endocr. Metab. Disord. 4:61-77, 2003). The preliminary results indicate that 4-months of leptin replacement was effective in reversing NASH in these patients in addition to causing significant improvements in metabolic parameters. Treatment with recombinant leptin has also been highly effective in the treatment of morbid obesity associated with congenital leptin deficiency. These patients also display a number of hormonal and metabolic abnormalities which respond to leptin therapy. To our knowledge, the liver histopathology that is associated with this human condition has not been described. Thus, whether leptin therapy leads to a beneficial therapeutic effect in the livers of these rare patients is not known.
While leptin therapy leads to a number of beneficial effects in conditions associated with leptin deficiency, the majority of human obesity is thought to be associated with leptin resistance. In rodents, leptin appears to exert a therapeutic effect in models of leptin resistance when sufficient amounts of the hormone are administered (Halaas et al., Proc. Natl. Acad. Sci. U.S.A. 94:8878-8883, 1997). In addition, central administration of leptin results in beneficial effects in many of the models of obesity (Halaas et al., supra). These observations had triggered significant enthusiasm for the development of recombinant leptin as a therapy for obesity. However, the obesity trials in humans indicated leptin resistance to be a more significant problem than in rodents. In a Phase II trial, obese subjects treated with leptin did not demonstrate statistically different amounts of weight loss compared to the placebo-treated subjects. Careful analyses of the presented data indicates the presence of at least 25% of patients with clinically meaningful and powerful responses producing weight loss of up to 16 kg (Heymsfield et al., JAMA 282:1568-1575, 1999). The parameters that distinguish responders from non-responders were not published. Since leptin has been clinically effective in states of leptin deficiency, one plausible hypothesis would be that the responders had relatively lower levels of leptin at baseline, indicating higher leptin sensitivity. However, such an analysis has not been provided from the Phase II trial. The studies exploring leptin's therapeutic potential in obesity did not evaluate the efficacy on liver histopathology. In addition, patients with abnormal liver function were specifically excluded from those studies.
As it is clear from evidence presented thus far, the question of leptin's role in the development of common forms of NASH in humans is not yet answered. The first step in determining the answer to this question is to determine what leptin levels are in NASH. The consensus from a handful of reports investigating this question is that leptin levels appear to be “elevated” as a mean compared to “healthy” normals (Chitturi et al., Hepatology 36:403-409, 2002; Uygun et al., Am. J. Gastroenterol. 95:3584-3589, 2000; Giannini et al., Hepatogastroenterology 46:2422-2425, 1999; Chalasani et al., Am. J. Gastroenterol. 98:2771-2776, 2003). However, it is hard to reach a conclusion from these reports for various reasons. First of all, two of these studies were done outside the United States and the leptin levels of the “control groups” were lower than the population normals published in the United States (Uygun et al., supra; Giannini et al., supra). Hence, their applicability or validity for the patients in the United States is unclear. Further, these studies, except for one (Chalasani et al, supra) did not evaluate regional body composition in terms of subcutaneous and visceral adipose tissue compartments (Chitturi et al., supra; Uygun et al., supra; Giannini et al., supra). Insulin resistance was not evaluated in relationship to body composition and leptin levels (Chitturi et al., supra; Uygun et al., supra; Giannini et al., supra). The differences between different BMI categories were not explored (Chitturi et al., supra; Uygun et al., supra; Giannini et al., supra). As indicated in the most recent paper (Chalasani et al, supra), the number of patients were limited, and one of the studies had only six patients with NASH (Giannini et al., supra). Comparisons of circulating leptin levels in patients with NASH with a so-called control group has been misleading since it is impossible to define a true control group without performing a liver biopsy (Chalasani et al, supra). Most importantly, leptin levels were not analyzed as the variable affecting other characteristics.
While studying and interpreting leptin levels, it is important to recognize that circulating leptin levels are a function of total body adiposity. Large epidemiological studies suggest that leptin levels correlate with body weight and BMI (Considine et al., N. Engl. J. Med. 334:292-295, 1996; Takahashi et al., Horm. Metab. Res. 28:751-752, 1996). The characteristics of body fat depots also play a contributory role in determining leptin levels. For example, subcutaneous fat is a more efficient producer of leptin (Takahashi et al., supra).
Based on the data from lipodystrophic patients and rodents collectively, it is hypothesized that circulating leptin level is an important signal in the modulation of the pathophysiology of lipid deposition in the liver. It is further hypothesized that a low leptin level plays an important role in communicating to the liver whether this organ should be utilized as a lipid storage site. When the storage capacity of adipose tissue is inherently low (which is associated with low leptin levels) or is beyond maximum capacity (associated with high leptin levels, but there is a state of leptin resistance), the liver takes on an additional role of “energy storage” and becomes engorged with fat. Fat deposition in humans makes the liver vulnerable to metabolic injury. This vulnerability develops into the clinical picture of NASH. In this paradigm, it is hypothesized that the patients with low leptin levels will respond to exogenous leptin therapy aiming to augment their leptin levels.
Leptin has clear anti-steatotic effects. The exact mechanisms of leptin's anti-steatotic effects in various tissues are not known. To elucidate the mechanism by which leptin reduces hepatic lipid content, Cohen et al, used microarrays to identify genes in liver that were differentially regulated by leptin or by food restriction (pair-feeding) (Cohen et al., Science 297:240-243, 2002). Leptin-treated ob/ob mice lose significantly more weight than pair-fed ob/ob mice, indicating that leptin stimulates energy expenditure. The authors identified 15 clusters of genes with distinct patterns of expression, six of which correspond to genes specifically regulated by leptin, but not by pair-feeding. To prioritize leptin-regulated genes for functional analysis, the authors then developed an algorithm to identify and rank genes that are specifically repressed by leptin. The gene encoding stearoyl-CoA desaturase (SCD-1) ranked the highest in this analysis. The microsomal enzyme SCD-1 is required for the biosynthesis of the monounsaturated fats palmitoleate and oleate from saturated fatty acids (Ntambi, J. Lipid Res. 40:1549-1558, 1999). SCD-1 RNA levels were markedly elevated in untreated ob/ob liver and these levels normalized with leptin treatment. Pair-fed mice showed a smaller and delayed decrease in SCD-1 gene expression. Also consistent with this observation, the authors noted that mice which carry mutations in SCD-1 had significantly reduced body fat mass. Furthermore, crossing leptin deficient ob/ob mice with the SCD-1 deficient mice produced a marked decrease in hepatic lipid content, size of the liver and a marked reduction in circulating serum triglyceride concentrations, effects similar to leptin treatment. These observations suggest that a significant proportion of leptin's metabolic effects may result from inhibition of SCD-1. Additionally, leptin increases hepatic and adipocyte expression of PPARγ coactivator (PGC)-1α 33, which regulates mitochondria biogenesis and fat oxidation (Wu et al., Cell 98:115-124, 1999).
Another important anti-steatotic effect of leptin was uncovered by studying muscle tissue. Minokoshi and colleagues showed that leptin selectively stimulates phosphorylation and activation of the α2 catalytic subunit of 5′AMP-activated protein kinase (AMPK) (α2 AMPK) in skeletal muscle, thus establishing a previously unknown signaling pathway for leptin (Minokoshi et al., Biochem. Soc. Trans. 31:196-201, 2003). In parallel with its activation of AMPK, leptin suppresses the activity of acetyl CoA carboxylase (ACC), thereby stimulating the oxidation of fatty acids in muscle. Whether leptin causes its peripheral effects via its central action or directly through its peripheral receptors is not known. For instance, the lipopenic action of leptin has been duplicated in rodents with small, centrally administered doses (Asilmaz et al., J. Clin. Invest. 113:414-424, 2004). The effect is achieved even with the knockout of peripheral leptin receptors (Cohen et al., J. Clin. Invest. 108:1113-1121, 2001), suggesting that leptin signals through one or more centrally-mediated intermediates, which in turn lead to increased fatty acid oxidation and suppression of lipogenesis.
Currently there are no good clinical markers that allow for the identification of patients with NASH. Similarly, there are no therapies to slow down or alter the course of further disease progression in NASH. Such markers and treatment for NASH are needed in the art. NASH ranks as one of the major causes of cirrhosis in America, behind hepatitis C and alcoholic liver disease. Thus, there exists a need in the art for methods of treating NASH. The invention provides recombinant leptin therapy for patients with NASH who demonstrate a leptin deficiency, i.e., low levels of circulating leptin. The invention also provides insight into the mechanisms by which leptin may regulate the pathology of NASH. The invention also provides insight into understanding the link between NASH and obesity, focusing on a potential mechanism that regulates fat deposition in the body. This mechanism involves the adipocyte hormone leptin which plays a key role in determining the status of energy availability and energy partitioning. Leptin has strong anti-steatotic effects proven in rodents and in rare human conditions such as in lipodystrophy syndromes.